Interferons are a family of proteins that occur in a variety of vertebrates and that have numerous activities, including the regulation of certain responses to disease. The mechanisms by which interferon blocks virus replication remain unclear. Double-stranded RNA (dsRNA) formed during virus replication has been implicated in this process; it may activate two interferon-induced proteins, 2-5A synthetase and protein P1 kinase, that result in RNA degradation and translational inhibition, respectively. The presence of dsRNA in virus-infected cells has not been adequately investigated; most previous investigations utilized extraction procedures that probably resulted in artifactual base pair formation. Furthermore, the effects of interferon on dsRNA formation have not been well catalogued. The proposed research will investigate dsRNA in intact cells through employment of specific nucleic acid antibodies and psoralen cross-linking techniques. The effect of cross-linking an antibody recognition will be evaluated and, if possible, antibodies specific for cross-linked dsRNA will be made utilizing hybridoma technology if necessary. A sensitive assay for the quantitation of low levels of dsRNA will be developed. DsRNA in interferon-treated or control mouse L cells infected with encephalomyocarditis virus will be quantitated as a function of the multiplicity of infection, the time post infection and the time and dose of interferon pretreatment. DsRNA containing material will be isolated wihout exposure to harsh denaturants. This material will be characterized with respect to the number and nature of the RNA and proteins(s) present. This research will further our understanding of dsRNA and the antiviral action of interferon, and will result in valuable reagents useful to approach other questions regarding the significance of dsRNA in the action of interferon and in various disease states.